Adenylosuccinate lyase (ASL)
catalyzes 2 reactions in the de novo synthesis of purine nucleotides. In
humans, point mutations of this enzyme are associated with mental retardation,
autism, and muscle wasting. The sequence of B. subtilis ASL is 27% identical
and 17% similar to the human enzyme. We have cloned, expressed, and purified
B. subtilis ASL; the crystal structure of a 73% homologous bacterial enzyme
is known (PDB #lc3u). We separately mutated 2 amino acids in the B. subtilis
ASL (M10L & N276C) equivalent to the mutations identified in human
ASL deficiency (M26L & R303C). The purified M10L and N276C possess
specific activities of 0.86 and 0.38 units/mg, respectively, as compared
to 1.56 units/mg for wild type (WT) enzyme. Both enzymes are similar to
WT in conformation and oligomeric state as indicated by circular dichroism
and light scattering. The Km for adenylosuccinate (SAMP) is not appreciably
different between M1OL (5.6 uM) and WT (4.9 uM); and the pH dependence
of Vmax is also similar. However, a marked difference between the 2 enzymes
is observed in the thermal denaturation experiments: at 42.5 °C, M1OL
enzyme rapidly loses activity with k = 0.08 min-1. WT enzyme
is completely stable over 1 hour. The instability of the human equivalent
of the M1OL enzyme may account for ASL deficiency in patients. In contrast.,
the N276C enzyme is as stable as WT enzyme at 42.5 °C. In N276C, the
Km for SAMP is increased 2-fold at pH 7.0, and up to 4-5 fold at pH 8.0-8.5,
compared to WT enzyme. The reduced activity of N276C may be attributed
to changes in the pH dependence of KM for SAMP. Replacement of N276 may
distort the adjacent E275, which is normally 2.7 A from H141 in the active
site and influences the pK of this criticaI general acid/ general base
as well as affecting substrate binding. Distortion of the E275-H141 interaction
caused by change of the human equivalent of position 276 may be the basis
of ASL deficiency in these patients. (Supported by NSF Grant MCB-97-28202).

Eristostatin, a disintegrin
found in viper venom, can inhibit the metastasis of melanoma cells in a
mouse model. To identify the amino acids that are critical for eristostatinís
interaction with melanoma cells, we expressed bacterial recombinant eristostatin,
echistatin (a structurally similar disintegrin), and six mutations targeting
amino acids known to be important for these disintegrinsí biological activities.
Human 1205 Lu, 164, and C8161 melanoma cells were incubated in an eight-chambered
slide in the presence of fluorescently labeled eristostatin and 0, 500,
or 1000 nM of an unlabeled disintegrin. Human MV3 melanoma cells were incubated
with fluorescently labeled anti-?4 and 3000 nM unlabeled disintegrin. The
cells were observed by confocal microscopy. For 1205 Lu, 164, and C8161
cells, the mutations in which RGD had been replaced with AGD or from which
the six C-terminal residues had been removed did not inhibit labeled eristostatinís
binding to the cells. This suggests that the RGD sequence and the C-terminus
are critical for eristostatinís binding to these cells. Four proteins with
methionine at position RGDXM inhibited labeled eristostatinís binding at
a higher level than unlabeled eristostatin did. This suggests that asparagine
is not a critical residue and that methionine enhances the ability of the
disintegrin to interact with 1205 Lu, 164, and C8161 cells. Eristostatinís
interaction with MV3 cells, similar to the other three cell lines, involves
the RGD sequence and the C-terminus. Eristostatin did not inhibit when
asparagine at position 31 or tryptophan at position 30 were replaced, suggesting
that those two residues are critical for eristostatinís binding to MV3
cells. We hypothesize that eristostatin uses the same mechanism to interact
with 1205 Lu, 164, and C8161 cells, and that this mechanism is different
than the one used with MV3 cells.

Anhydrotic Ectodermal Dysplasia
(EDA) is a genetic disorder, which is X-linked and recessive. Individuals
affected with this disorder experience dysfunctional sweat glands, poorly
developed teeth, and premature balding. The gene affected by this disorder
is Ectodysplasin, a protein that is proposed to be involved in cellular
signaling. Mice with mutations in Ectodysplasin develop the Tabby phenotype.
These mice have defects similar to humans with EDA, and in addition, develop
a kink in their tails. The phenotype of the Tabby tails was examined via
a whole-mount stain involving Alizarin Red and Alician Blue, and the histology
was examined by hemotoxylin and eosin staining. The structural integrity
of the bone was examined through 3-dimensional microcomputed tomography,
focussing on the tibia. Using statistical analysis, it was determined that
the trabecular bone characteristics of the tibia differed between the Tabby
mutant and the wild type mouse. Trabecular bone volume and trabecular connections
were greater for the Tabby mutant than for the wild type. Trabecular separation
was decreased for the Tabby when compared to the wild type. Since it has
been recently determined that Ectodysplasin is expressed in the bone of
developing mouse embryos, this evidence suggests a taking a closer look
into the bone integrity of individuals affected with EDA

In E. coli, IscS
and Thil are the only proteins needed to convert the uridine at position
8 of tRNA into 4-thiouridine (s4U)í. If E. coli are subject
to near-UV light, a crosslink forms between the s4U at position
8 and the cytidine at position 13 on the same tRNA. This crosslink makes
tRNA a poor substrates for amino-acyl tRNA synthetases, therefore causing
arrested cell growth2. Eukaryotes lack s4U in tRNA and Thu homologs,
but eukaryotes do have IscS homologs. Since unfractionated yeast tRNA is
a susbstrate for E.coli enzymes in vitro, we reasoned that expression
of Thu in yeast should lead to s4U generation in S. cerevisiae,
which would prove a useful system for both revealing substrate specificity
and monitoring gene expression in yeast. A vector appropriate for expression
of Thu in yeast has been constructed and transformed into S. cerevisiae.
The content of s4U in isolated yeast tRNA will be monitored
before and after induction of Thu expression, and if s4U is
observed when Thu is expressed, the sensitivity of the yeast to near-UV
light will be examined. To confirm that Thil and the yeast IscS homolog,
Nfs1p, can function together, NSF1 has been cloned for overexpression
in E. coli, and the purified yeast protein will be assayed for ability
to support in vitro s4U production in conjugation with ThIL
The results of these experiments will be presented.1. Kampabati R, Lauhon
CT. 1999. IscS is a sulfurtransferase for the in vitro biosynthesis of
4-thiouridine in Escherichia coli tRNA. Biochemistry 38:16561-16568.2. Favre, A. Bioinorganic
Chemistry: Photochemistry and the Nucleic Acids. New York: John Wiley and
Sons. 1990

Apolipoprotein C-Ill (apoC-Ill),
a protein which influences the catabolism of triglyceride-rich lipoproteins,
has previously been observed only in mammals. A cDNA sequence was isolated
from a turtle, Pseudymes scripta, liver expression library, and shown to
be the equivalent to mammalian apoC-III. Comparison of the turtle and human
sequences indicated 39% amino acid identity but 61% similarity. In particular,
sequences for the second and third of the six 11 amino acid (11-mer) repeats
were highly conserved. Like mammalian apoC-III, turtle apoC-Ill with a
predicted MW of 10,504, is found associated with both HDL and triglyceride-rich
lipoproteins and is synthesized mainly by the liver. Using PCR primers
based on the cDNA sequence, the sequence of the turtle apoC-Ill gene was
determined. Like mammalian apoC-III, the turtle gene contains four exons
and three introns. The exon length and exon/intron boundries are also similar
to those found for humans although the introns are more variable. The results
suggest that exon 3 which encodes the first, second and third 11-mer repeats
has been more highly conserved relative to the fourth exon which encodes
the rest of the protein. The conservation of the cDNA sequence, gene structure
and lipoprotein associations suggests turtle apoC-III plays a role similar
to that described in mammals. Genome walking was then used to establish
that the turtle apoC-Ill and apoA-I genes were linked. As in mammals, the
orientation of the two genes is opposing, with the promoters for the two
genes being distal and the 3í ends being proximal to each other. Since
the evolution of turtles is separated by an estimated 300 millions years
from humans, the conservation of the linkage group supports the conclusion
that the two genes play a coordinate role in lipoprotein metabolism.

Chimeric oligonucleotides
consisting of both RNA and DNA configured as a double-hairpin have been
shown to cause site-directed mutations involving the processes of homologous
recombination and gene conversion. In order to increase the efficiency
of gene repair, we have been studying the mechanism of recombination with
both prokaryotic and eukaryotic recombinases. To measure the relative efficiencies
of gene targeting we have adapted an in vitro system, involving the incubation
of various model RNA/DNA hybrid hairpins and recombinases, followed by
the addition of a radio-labeled target. After stripping away the recombinase,
the resulting chimera/target joints were analyzed by PAGE. Recombination,
mediated by eukaryotic Rad5 1 protein, produced a low yield of Y-arm product
formation with short linear dsDNA targets. Although the efficiency of the
product formation is considerably less than that observed for the same
system using RecA, we suspect using accessory proteins such as Rad54 will
dramatically increase targeting efficiency. To our knowledge this is the
first demonstration of reciprocal four-strand exchange catalyzed by the
Rad5 1 protein. On the other hand RecA mediated recombination produced
high levels of double D-loop product with both short linear dsDNA and supercoiled
plasmid targets. The formation of double D-Ioops under these conditions
approached 90%, far more efficient than what has been reported previously.
Earlier research suggested the formation of a double D-loop intermediate
during gene targeting; these results give ftirther evidence to support
this conclusion, as well as a method of efficiently producing these structures
for further mechanistic studies.